siRNA Targeting VEGFA and Methods for Treatment In Vivo

ABSTRACT

Vascular endothelial growth factor A (VEGFA) is a chemical signal produced by cells that stimulates the growth of new blood vessels, and overexpression of VEGFA can lead to undesirable physiological conditions. Through the identification of new siRNA and modifications that improve the silencing ability of these siRNA in vivo, therapeutic compositions and methods have been invented to address the problems associated with this overexpression.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 61/368,385, filed Jul. 28, 2010, theentire disclosure of which is incorporated by reference as if set forthfully herein.

FIELD OF INVENTION

The present invention relates to the use of siRNA.

BACKGROUND OF THE INVENTION

Angiogeneis is a physiological process that involves the growth of newblood vessels. An important part of this process is the production ofvascular endothelial growth factor (“VEGF” or “VEGFA”), which is achemical signal that is produced by cells and that stimulates the growthof new blood vessels.

The process is initiated when VEGFA is secreted by cells and binds toone or more cognate receptors such as the transmembrane protein kinaseVEGFR1/FLT-1 and VEGFR2/FLK-1/KDR. After VEGFA binds to thetransmembrane protein, a signal cascade is initiated that ultimatelyresults in neovascularization.

Angiogeneis can be part of normal and vital body development andregulation. Unfortunately, it can also be associated with a number ofundesirable conditions such as retinopathy, psoriasis, cancer, exudativeage-related macular degeneration (ARMD), and rheumatoid arthritis. Inthese conditions, as well as in others, there are both high levels ofVEGFA and concomitant increases in vascularization. Thus, thedevelopment of therapeutic strategies that focus on control of theproduction of VEGFA are being sought.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods for thesuppression of VEGFA expression, as well as to the treatment ofconditions that are associated with the overexpression of VEGFA.Accordingly, the present invention provides kits, siRNAs and methods forintroducing siRNA that suppress, in whole or in part, the production ofVEGFA.

According to a first embodiment, the present invention provides a methodfor suppressing the expression of VEGFA. The method comprisesadministering in vivo (e.g., in a human) an siRNA that comprises asequence that is a selected from the group consisting of: SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, and 88 to an organism.

According to a second embodiment, the present invention provides amethod for suppressing the expression of VEGFA. The method comprisesadministering in vitro an siRNA that comprises a sequence that isselected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, and 88.

According to a third embodiment, the present invention provides a methodfor suppressing expression of VEGFA. The method comprises administeringan siRNA according to either of the first two embodiments, wherein thesiRNA has one or more of the following modifications: 2′-O-alkyl (e.g.,2′-O-methyl) modifications of all C and U nucleotides within the sensestrand as well as on the first two 5′ nucleotides of the sense strand,2′ Fluoro modifications of all of the C and U nucleotides within theantisense strand and a 5′ phosphorylation of the nucleotide at positionone of the antisense strand. In some embodiments the siRNA has2′-O-alkyl modifications on all C and U nucleotides within the sensestrand and at least one 2′-O-alkyl modification on the antisense strand.In some embodiments the siRNA has one or more overhangs of one to sixnucleotides. In some embodiments all of the aforementioned modificationsare present, and only those modifications are present, thus, all G and Anucleotides, other than those located at positions 1 and 2 of the sensestrand have 2′-OH groups.

According to a fourth embodiment, the present invention provides amethod for suppressing expression of VEGFA. The method comprisesadministering an siRNA according to any of the first three embodiments,wherein the siRNA has one or more of the following modifications: acholesterol moiety attached by a C5 linker, and mismatches at one ormore of positions 6, 13 and 19 of the sense strand where the sensestrand is 19 nucleotides long and the antisense strand is also 19nucleotides in length (excluding overhangs). The positions on the sensestrand are measured from the 5′ end of the sense strand wherein thefirst 5′ nucleotide of the sense strand is identified as the 5′-mostnucleotide that is base-paired with a nucleotide on the antisensestrand. As such, by this definition, 5′ sense strand overhangnucleotides are not included in the counting scheme. In some of theseembodiments there are no 5′ overhangs. In some of the embodiments thereare one or two 3′ overhangs of 1 to 6 bases or there are no overhangs.In some embodiments, except for at positions 6, 13 and 19, within theduplex region, there is 100% complementarity.

According to a fifth embodiment, the present invention provides a poolof at least two siRNA selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86 and 88.

According to a sixth embodiment, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof one or more of the siRNAs disclosed herein.

According to a seventh embodiment, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof an siRNA, wherein the siRNA consists of: (a) an antisense strand thatis nineteen to thirty-six bases in length and that comprises a sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, and 88; and (b) a sense strand that is nineteen to thirty-six basesin length, wherein the antisense strand and the sense strand form aduplex region of seventeen to thirty base pairs and within the duplexregion there is at least 75% complementary. In the event that the duplexregion of the siRNA is longer than 19 base pairs in length, additional(sense and antisense) sequences are added to the 3′ end of the antisensestrand and 5′ end of the sense strand.

Through the use of the methods, siRNAs and pharmaceutical compositionsdescribed herein, one may efficiently and effectively silence VEGFA.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B demonstrate the in vivo silencing activities of two VEGFAsiRNAs modified as Accell molecules and delivered by intravitreal (IVT)injection in rats.

FIG. 2 is a representation of a dose response curve for an siRNA, VEGFA3.2 (modified as an Accell molecule).

FIGS. 3A and 3B are representations of dose response curves for anothersiRNA, VEGFA 3.7 (modified as an Accell molecule).

FIGS. 4A and 4B demonstrate a duration of action of up to eight weeksfor an siRNA, VEGFA 3.7, modified as an Accell molecule and delivered byIVT injection in rats.

FIGS. 5A and 5B demonstrate inhibition of VEGFA expression andpreretinal neovascularization in the rat oxygen-induced retinopathy(OIR) model by siRNA, VEGFA 3.7, modified as an Accell molecule.

DETAILED DESCRIPTION Definitions

Unless stated otherwise or apparent from context, the following termsand phrases have the meanings provided below:

2′ Modification

A 2′ modification refers to a substitution of the hydroxyl group that istypically located at the 2′ position of a ribose sugar within aribonucleotide, with another moiety, e.g., an —O-alkyl group such as—O-methyl, —O-ethyl, —O-n-propyl, —O-isopropyl etc., or another groupsuch as a fluoro group. Where —O-alkyl modifications are present, insome embodiments the same —O-alkyl group is present on allO-alkyl-modified nucleotides. Other types of 2′ modifications arehalogen groups, e.g., 2′ Fluoro, or 2′ bromo.

“Accell” siRNA

The term “Accell” refers to a preferred siRNA structure comprising thefollowing: the sense strand is 19 nucleotides long and has: (1)2′-O-methyl modifications on positions 1 and 2 (counting from the 5′terminus); (2) 2′-O-methyl modifications on all Cs and Us; and (3)cholesterol conjugated to the 3′ terminus via a C5 linker. The antisensestrand is 21 nucleotides in length, has a 5′ phosphate modification,contains a 2′ F modification on all Cs and Us, forms a 2 nucleotideoverhang when paired with the sense strand, and contains phosphorthioatemodification between (1) the two nucleotides of the overhang, and (2)between the 3′ most nucleotide of the duplexed region and the firstnucleotide of the overhang. In addition, Accell molecules containmismatches at positions 6, 13, and 19 (counting from the 5′ end of thesense strand). In all cases, these mismatches are generated by replacingthe sense nucleotide with an alternative base. In this way, theantisense strand retains complete complementarity with the targetmolecule. For additional details, see US 2009/0209626 A1, the disclosureof which is incorporated by reference.

Complementary

The term “complementary” refers to the ability of polynucleotides toform base pairs with one another. Base pairs are typically formed byhydrogen bonds between nucleotide units in antiparallel polynucleotidestrands. Complementary polynucleotide strands can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenosine.However, when a U is denoted in the context of the present invention,the ability to substitute a T is implied, unless otherwise stated.

Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can hydrogen bond with each other.For example, for two 20-mers, if only two base pairs on each strand canhydrogen bond with each other, the polynucleotide strands exhibit 10%complementarity. In the same example, if 18 base pairs on each strandcan hydrogen bond with each other, the polynucleotide strands exhibit90% complementarity. In some embodiments, within a duplex region, thereis at least 75% complementarity, at least 80% complementarity, at least90% complementarity, at least 95% complementarity or 100%complementarity.

Conjugate Moiety

Conjugate moieties of the disclosure (also referred to simply as“conjugates”) are moieties that are connected either directly orindirectly to a nucleotide and can target entry into a cell by a varietyof means. For instance, conjugate moieties can be lipid in nature. Assuch, lipid based conjugate moieties can include cationic lipids,neutral lipids, sphingolipids, and fatty acids including stearic, oleic,elaidic, linoleic, linoleaidic, linolenic, and myristic acids.Alternatively, the conjugate moieties can be proteinaceous in natureincluding peptides that are membrane translocating (e.g., TAT,penetratin, MAP) or cationic (e.g., poly(lys), poly(arg), poly(his),poly (lys/arg/his), or protamine).

Alternatively, the conjugate moiety can be a small molecule that, forinstance, targets a particular receptor or is capable of insertingitself into the membrane and being absorbed by endocytic pathways. Thus,small molecules based on adamantanes, polyaromatic hydrocarbons (e.g.,napthalenes, phenanthrenes, or pyrenes), macrocyles, steroids, or otherchemical scaffolds, are all potential conjugates for the disclosure.

In yet another alternative, conjugate moieties can be based on cationicpolymers, such as polyethyleneimine, dendrimers, poly(alkylpyridinium)salts, or cationic albumin.

In some cases, the conjugate moieties are ligands for receptors or canassociate with molecules that in turn associate with receptors. Includedin this class are bile acids, small molecule drug ligands, vitamins,aptamers, carbohydrates, peptides (including but not limited tohormones, proteins, protein fragments, antibodies or antibodyfragments),viral proteins (e.g., capsids), toxins (e.g., bacterialtoxins), and more. Also included are conjugates that are steroidal innature e.g., cholesterol, cholestanol, cholanic acid, stigmasterol,pregnelone, progesterones, corticosterones, aldosterones, testosterones,estradiols, ergosterols, and more. Preferred conjugate moieties of thedisclosure are cholesterol (CHOL), cholestanol (CHLN), cholanic acid(CHLA), stigmasterol (STIG), and ergosterol (ERGO).

In yet another embodiment, the molecules that target a particularreceptor are modified to eliminate the possible loss of conjugatedsiRNAs to other sources. For instance, when cholesterol-conjugatedsiRNAs are placed in the presence of normal serum, a significantfraction of this material will associate with the albumin and/or otherproteins in the serum, thus making the siRNA unavailable for e.g.,interactions with LDLs. For this reason, the conjugate moieties of thedisclosure can be modified in such a way that they continue to bind orassociate with their intended target (e.g., LDLs) but have lesseraffinities with unintended binding partners (e.g., serum albumin).

Duplex Region

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary polynucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for a stabilized duplex between polynucleotide strands thatare complementary or substantially complementary.

Examples of sizes of duplex regions include but are not limited to 17-30base pairs, 17-25 base pairs, 17-23 base pairs, 18-30 base pairs, 18-25base pairs, 18-23 base pairs, 19-30 base pairs, 19-25 base pairs and19-23 base pairs. A duplex region may be defined by the length of basepairs, as well as the degree of complementarity over that range.

Thus, when the duplex region is formed from two separate strands ofnucleotides, the antisense strand and the sense strand, it is importantto note that each strand may contain nucleotides that are part of theduplex and nucleotides that are not part of the duplex at either the 5′end or the 3′ end. An siRNA may be designed such that on the antisensestrand, all nucleotides that are complementary to a target are part ofthe duplex region, and thus have complementary nucleotides on the sensestrand. However, the siRNA may be also be designed such that theantisense strand also contains nucleotides at either its 3′ end and/orits 5′ end that although not having complementary nucleotides on thesense strand, are part of a continuous stretch of nucleotides within theantisense strand that have complementary nucleotides on the target.

By way of example, a sense strand may contain 19 nucleotides and anantisense strand may contain 21 nucleotides. All but the two 3′ mostnucleotides of the antisense strand may be complementary to the 19nucleotides on the sense strand, while the entire stretch of 21nucleotides of the antisense strand may be complementary to a stretch of21 nucleotides of the target. Alternatively, the two 3′ most nucleotidesof the antisense strand may be selected so as not to be complementary toa portion of the target, or selected randomly or to facilitateprocessing such that one or both might or might not be complementary tothe two nucleotides of the target that are adjacent to the nucleotidesto which the other 19 nucleotides of the antisense strand arecomplementary.

Additionally, in different embodiments, within a duplex region there mayfor example be no mismatches, one mismatch, two mismatches, threemismatches, four mismatches, or five mismatches.

Mismatch

The term “mismatch” includes a situation in which Watson-Crick basepairing does not take place between a nucleotide of a sense strand and anucleotide of an antisense strand. Examples of mismatches include butare not limited to an A across from a G, a C across from an A, a Uacross from a C, a U across from a G, an A across from an A, a G acrossfrom a G, a C across from C, and a U across from a U.

Linker

A linker is a moiety that attaches two or more other moieties. Thoughnot wishing to be limited by definitions or conventions, in thisapplication the length of the linker is described by counting the numberof atoms that represents the shortest distance between the atom thatjoins the conjugate moiety to the linker and the oxygen atom of theterminal phosphate moiety associated with the oligonucleotide throughwhich the linker is attached to the oligonucleotide. For example, inembodiments where the conjugate moiety is joined to the linker via acarbamate linkage, the length of the linker is described as the numberof atoms that represents the shortest distance between the nitrogen atomof the carbamate linkage and the oxygen atom of the phosphate linkage.In cases where ring structures are present, counting the atoms aroundthe ring that represent the shortest path is preferred.

Non-limiting examples of structures of the conjugate-linker that may beused in the compositions and methods of the disclosure include but arenot limited to linkers/linker chemistries that are based onβ-amino-1,3-diols, β-amino-1,2-diols, hydroxyprolinols,ω-amino-alkanols, diethanolamines, β-hydroxy-1,3-diols,β-hydroxy-1,2-diols, β-thio-1,3-diols, β-thio-1,2-diols,β-carboxy-1,3-diols, β-carboxy-1,2-diols, ω-hydroxy-alkanols,ω-thio-alkanols, ω-carboxy-alkanols, functionalized oligoethyleneglycols, allyl amine, acrylic acid, allyl alcohol, propargyl amine, andpropargyl alcohol.

In some embodiments a linker not only provides a site of attachment tothe conjugate moiety, but also provides functional sites for attachmentto the support and for initiation of oligonucleotide synthesis.Preferably, these sites are hydroxyl groups; most preferably, they are aprimary hydroxyl group and a secondary hydroxyl group, to allow them tobe chemically distinguished during synthesis of the conjugate-modifiedsolid support. One hydroxyl group, preferably the primary hydroxylgroup, is protected with a protecting group that can be removed as thefirst step in the synthesis of the oligonucleotide, according to methodswell understood by those of ordinary skill in the art. Preferably, thisprotecting group is chromophoric and can be used to estimate the amountof the conjugate moiety attached to the solid support; most preferably,the group is chosen from triphenylmethyl (Tr),monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMTr) andtrimethoxytriphenylmethyl (TMTr). Another hydroxyl group, preferably asecondary hydroxyl group, is derivatized with a functionalized tetherthat can covalently react with a functional group on the solid synthesissupport, according to methods well understood by those of ordinary skillin the art. Preferable tethers are, by way of example, dicarboxylicacids such as succinic, glutaric, terephthalic, oxalic, diglycolic, andhydroquinone-0,0′-diacetic. One of the carboxylic acid functionalitiesof the tether is reacted with the hydroxyl to provide an ester linkagethat is cleavable using basic reagents (hydroxide, carbonate or amines),while the other carboxylic acid functionality is reacted with thesynthesis support, usually through formation of an amide bond with anamine functionality on the support. The linker may also confer otherdesirable properties on the oligonucleotide conjugate: improved aqueoussolubility, optimal distance of separation between the conjugate moietyand the oligonucleotide, flexibility (or lack thereof), specificorientation, branching, and others.

Preferably, the chemical bond between the linker and the conjugatemoiety is a carbamate linkage; however, alternative chemistries are alsowithin the scope of the disclosure. Examples of functional groups onlinkers that form a chemical bond with a conjugate moiety include, butare not limited to, hydroxyl, amine, carboxylic acid, carboxylic acidhalide, carboxylic acid active ester, carbonyl, chlorocarbonyl,imidazolylcarbonyl, thiol, maleimide, haloalkyl, sulfonyl, allyl andpropargyl. Examples of chemical bonds that are formed between a linkerand a conjugate include, but are not limited to, those based oncarbamates, ethers, esters, amides, disulfides, thioethers,phosphodiesters, phosphorothioates, phorphorodithioate, sulfonamides,sulfonates, sulfones, sulfoxides, ureas, hydrazide, oxime, photolabilelinkages, C—C bond forming groups such as Diels-Alder cyclo-additionpairs or ring-closing metathesis pairs, and Michael reaction pairs. Ingeneral, the conjugate moiety will have an appropriate functional groupeither naturally or chemically installed; the linker will then besynthesized with a functional group chosen to efficiently and stablyreact with the functional group on the conjugate moiety.

Linkers that have the same length, but are capable of associating withtwo or more conjugates, are also specifically contemplated.

In another embodiment, the linker may be a nucleoside derivative. Thenucleoside may be, for example, a ribonucleoside,2′-deoxyribonucleoside, or 2′-modified-2′-deoxyribonucleoside, such as2′-O-methyl or 2′-fluoro. The nucleoside may be, for example, anarabinonucleoside or a 2′-modified arabinonucleoside. Using methods wellknown to those of ordinary skill in the art, purine and pyrimidinenucleosides may be modified at particular sites on the base to providelinkers and functional groups for attachment of conjugate moieties. Forexample, pyrimidine nucleosides, such as uridine and cytidine, may bemodified at the 5-position of the uracil or cytosine base using mercuricacetate, a palladium catalyst, and an allylic reagent such asallylamine, allyl alcohol, or acrylic acid. Alternatively,5-iodopyrimidines may be modified at the 5-position with a palladiumcatalyst and a propargylic reagent such as propargyl amine, propargylalcohol or propargylic acid. Alternatively, uridine may be modified atthe 4-position through activation with triazole or a sulfonyl chlorideand subsequent reaction with a diamine, amino alcohol or amino acid.Cytidine may be similarly modified at the 4-position by treatment withbisulfite and subsequent reaction with a diamine, amino alcohol or aminoacid. Purines may be likewise modified at the 7, 8 or 9 positions usingsimilar types of reaction sequences.

In preferred embodiments, the linker is from about 3 to about 9 atoms inlength. Thus, the linker may be 3, 4, 5, 6, 7, 8, or 9 atoms in length.Preferably, the linker is 5, 6, 7 or 8 atoms in length. More preferably,the linker is 5 or 8 atoms in length. Most preferably the linker is astraight chain C5 linker i.e., there are 5 carbon atoms between the atomthat joins the conjugate moiety to the linker and the oxygen atom of theterminal phosphate moiety associated with the oligonucleotide throughwhich the linker is attached to the oligonucleotide. Thus, where theconjugate moiety is joined to a C5 linker via a carbamate linkage, thereare 5 carbon atoms between the nitrogen atom of the carbamate linkageand the oxygen atom of the phosphate linkage.

In one preferred embodiment, the conjugate moiety is cholesterol and thelinker is a C5 linker (a 5 carbon linker) attached to the cholesterolvia a carbamate group, thus forming a Chol-C5 conjugate-linker. Whenattached via a phosphodiester linkage to the 5′ and/or 3′ terminus of asense and/or antisense oligonucleotide of a duplex, the resultingconjugate-linker-oligonucleotide can have the structure:

In another preferred embodiment, the conjugate moiety is cholesterol andthe linker is a C3 linker attached to the cholesterol via a carbamategroup, thus forming a Chol-C3 conjugate-linker. When attached via aphosphodiester linkage to the 5′ and/or 3′ terminus of a sense and/orantisense oligonucleotide, the resulting conjugatelinker-oligonucleotide can have the structure:

In another preferred embodiment, the conjugate moiety is cholesterol andthe linker is a C8 linker (a 8 carbon linker) attached to thecholesterol via a carbamate group, thus forming a Chol-C8conjugate-linker. When attached via a phosphodiester linkage to the 5′and/or 3′ terminus of a sense and/or antisense oligonucleotide, theresulting conjugate-linker oligonucleotide can have the structure:

In another preferred embodiment, the conjugate moiety is cholesterol andthe linker is a PRO linker (a 4 carbon linker) attached to thecholesterol via a carbamate group, thus forming a Chol-PROconjugate-linker.

In another preferred embodiment, the conjugate moiety is cholesterol andthe linker is a PIP linker (a 6 carbon linker) attached to thecholesterol via a carbamate group, thus forming a Chol-PIPconjugate-linker. When attached via a phosphodiester linkage to the 5′and/or 3′ terminus of a sense and/or antisense oligonucleotide, theresulting conjugate-linker-oligonucleotide can have the structure:

In another preferred embodiment, the conjugate moiety is cholesterol andthe linker is a C6-HP (also referred to as “HP6”) linker (a 9 carbonlinker) attached to the cholesterol via a carbamate group, thus forminga Chol-C6-HP conjugate-linker. When attached via a phosphodiesterlinkage to the 5′ and/or 3′ terminus of a sense and/or antisenseoligonucleotide, the resulting conjugatelinker-oligonucleotide can havethe structure:

Nucleotide

Unless otherwise specified, the term “nucleotide” refers to aribonucleotide or a deoxyribonucleotide or modified form thereof, aswell as an analog thereof. Nucleotides include species that comprisepurines, e.g., adenine, hypoxanthine, guanine, and their derivatives andanalogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, andtheir derivatives and analogs. In some emobodiments, all nucleotides areselected from the group of modified or unmodified A, C, G or U.

Nucleotide analogs include nucleotides having modifications in thechemical structure of the base, sugar and/or phosphate, including, butnot limited to, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at cytosine exocyclic amines, andsubstitution of 5-bromo-uracil; and 2′-position sugar modifications,including but not limited to, sugar-modified ribonucleotides in whichthe 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH₂,NHR, NR₂, or CN, wherein R is an alkyl moiety. Nucleotide analogs arealso meant to include nucleotides with bases such as inosine, queuosine,xanthine, sugars such as 2′-methyl ribose, non-natural phosphodiesterlinkages such as methylphosphonates, phosphorothioates and peptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications that cancomprise nucleotides that are modified with respect to the base moietiesinclude but are not limited to, alkylated, halogenated, thiolated,aminated, amidated, or acetylated bases, individually or in combination.More specific examples include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose,and other sugars, heterocycles, or carbocycles.

The term nucleotide is also meant to include what are known in the artas universal bases. By way of example, universal bases include but arenot limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term“nucleotide” is also meant to include the N3′ to P5′ phosphoramidate,resulting from the substitution of a ribosyl 3′ oxygen with an aminegroup.

Further, the term nucleotide also includes those species that have adetectable label, such as for example a radioactive or fluorescentmoiety, or mass label attached to the nucleotide.

Pharmaceutically Acceptable Carrier

The phrase “pharmaceutically acceptable carrier” means apharmaceutically acceptable salt, solvent, suspending agent or vehiclefor delivering a composition of the present disclosure to an organismsuch as an animal or human. The carrier may be liquid, semisolid orsolid, and is often synonymously used with diluents, excipient, or salt.The phrase “pharmaceutically acceptable” means that any ingredient,excipient, carrier, diluent or component disclosed is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, isolation and allergic response) commensuratewith a reasonable benefit/risk ratio. See Remington's PharmaceuticalScience 16^(th) edition, Osol, A. Ed. (1980).

Ribonucleotide and Ribonucleic Acid

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), referto a modified or unmodified nucleotide or polynucleotide comprising atleast one ribonucleotide unit. A ribonucleotide unit comprises anhydroxyl group attached to the 2′ position of a ribosyl moiety that hasa nitrogenous base attached in N-glycosidic linkage at the 1′ positionof a ribosyl moiety, and a moiety that either allows for linkage toanother nucleotide or precludes linkage.

Sense Strand/Antisense Strand

The phrase “sense strand” refers to a polynucleotide that comprises asequence that is in whole or in part, the same as a target nucleic acidsequence such as messenger RNA or a sequence of DNA. The phrase“antisense strand” refers to a polynucleotide that comprises a sequencethat is in whole or in part, the complement of a target nucleic acidsequence such as messenger RNA or a sequence of DNA.

When a sequence of an siRNA is provided, by convention, unless otherwiseindicated it is of the sense strand, and the complementary antisensestrand is implicit. In a duplex siRNA (formed from two separate strands)one strand may be the sense strand, and the other strand may be theantisense strand. If overhangs are present, the phrase “sense region”may refer to the nucleotide sequence portion of the sense strand otherthan overhang regions. Similarly, the phrase “antisense region” mayrefer to the nucleotide sequence portion of the antisense strand otherthan overhang regions. If the siRNA is a shRNA, there are not twoseparate strands, and the “sense region” is the portion of the duplexregion that has a sequence that is in whole or in part the same as thetarget sequence, and the “antisense region” is the sequence ofnucleotides that is in whole or in part complementary to the targetsequence and to the sense region.

Examples of lengths of sense strands and antisense strands are 19-36bases, 19-30 bases, 19-25 bases and 19-23 bases. These strand lengthsinclude possible overhang regions.

siRNA

The term “siRNA” refers to small inhibitory RNA duplexes that induce theRNA interference (RNAi) pathway. As used herein, these molecules canvary in length (generally 17-30 base pairs plus overhangs) and containvarying degrees of complementarity to their target mRNA in the antisensestrand. Some, but not all, siRNA have unpaired overhanging bases on the5′ or 3′ end of the sense strand and/or the antisense strand. The term“siRNA” includes duplexes of two separate strands, and unless otherwisespecified as well as single strands that can form hairpin structurescomprising a duplex region, which is referred to as a shRNA.

siStable

The term “siStable” refers to a chemical modification pattern that isassociated with a particular duplex. Specifically, siStable siRNAcomprise the following structures: the sense strand is 19 nucleotideslong and has (1) 2′-O-methyl modifications on positions 1 and 2(counting from the 5′ terminus), and (2) 2′-O-methyl modifications onall Cs and Us. The antisense strand is 21 nucleotides in length, has a5′ phosphate modification, contains a 2′ F modification on all Cs andUs, forms a 2 nucleotide overhang when paired with the sense strand, andcontains phosphorthioate modifications between (1) the two nucleotidesof the overhang, and (2) between the 3′ most nucleotide of the duplexedregion and the first nucleotide of the overhang. For details, see US2007/0269889 A1.

Target

The term “target” is used in a variety of different forms throughoutthis document and is defined by the context in which it is used. “TargetmRNA” refers to a messenger RNA to which a given siRNA can be directedagainst. “Target sequence” and “target site” refer to a sequence withinthe mRNA to which the sense strand of an siRNA shows varying degrees ofidentity and the antisense strand exhibits varying degrees ofcomplementarity. The phrase “siRNA target” can refer to the gene, mRNA,or protein against which an siRNA is directed. Similarly, “targetsilencing” can refer to the state of a gene, or the corresponding mRNAor protein.

Therapeutically Effective Amount

A “therapeutically effective amount” of a composition containing asequence that encodes a VEGFA-specific siRNA (i.e., an effectivedosage), is an amount that inhibits expression of the polypeptideencoded by the VEGFA target gene by at least 10 percent. Higherpercentages of inhibition, e.g., at least 15, at least 20, at least 30,at least 40, at least 50, at least 75, at least 85, at least 90 percentor higher may be preferred in certain embodiments. The skilled artisanwill appreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a composition can include a single treatment or a series oftreatments. In some cases transient expression of the siRNA may bedesired. When an inducible promoter is included in the constructencoding an siRNA, expression is assayed upon delivery to the subject ofan appropriate dose of the substance used to induce expression.

Appropriate doses of a composition depend upon the potency of themolecule (the sequence encoding the siRNA) with respect to theexpression or activity to be modulated. One or more of these moleculescan be administered to an animal (e.g., a mammal such as a human orother primate, e.g., a chimpanzee, orangutan, ape, monkey etc., or dog,cat, horse, cow, rat, sheep, or mouse) to modulate expression oractivity of one or more target polypeptides. A physician may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

A therapeutically effective amount of a VEGFA-specific siRNA is usefulfor treating a condition, disease, or disorder associated with elevatedexpression of VEGFA, including, but not limited to, psoriasis, cancer,rheumatoid arthritis, ocular neovascularization, abnormal angiogenesis,retinal vascular permeability, retinal edema, diabetic retinopathy(particularly proliferative diabetic retinopathy), diabetic macularedema, exudative age-related macular degeneration, sequela associatedwith retinal ischemia, and posterior segment neovascularization.

Preferred Embodiments

The present invention will now be described in connection with preferredembodiments. These embodiments are presented in order to aid in anunderstanding of the present invention and are not intended, and shouldnot be construed, to limit the invention in any way. All alternatives,modifications and equivalents that may become apparent to those ofordinary skill upon reading this disclosure are included within thespirit and scope of the present invention.

Furthermore, this disclosure is not a primer on compositions or methodsfor performing RNA interference. Basic concepts known to persons skilledin the art have not been set forth in detail.

According to a first embodiment, the present invention provides a methodfor decreasing expression of VEGFA, in vivo, comprising administering ansiRNA to an organism, wherein the siRNA comprises a sequence selectedfrom SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88.

The subject may be any organism that possesses an RNAi pathway,including, but not limited to a mammal, bird or reptile. Examples ofmammals include, but are not limited to humans, monkeys, apes,chimpanzees, dogs, cats, mice and rats.

In addition, the duplex formed by the sense strand and the antisensestrand can comprise at least one overhang, each overhang comprising atleast one nucleotide. The overhang(s) can for example be located:

at the 5′ end of the sense strand;

at the 3′ end of the sense strand;

at the 5′ and 3′ end of the sense strand;

at the 5′ end of the antisense strand;

at the 3′ end of the antisense strand;

at the 5′ and 3′ end of the antisense strand;

at the 5′ end of the sense strand and the 5′ end of the antisensestrand; or

at the 3′ end of the sense strand and the 3′ end of the antisensestrand.

In some embodiments, the overhang is six or fewer nucleotides in length,in preferred embodiments, an overhang is present at the 3′ end of theantisense strand, i.e., attached to the 3′ most nucleotides of theantisense regions. More preferably, the overhang on the 3′ end of theantisense strand is two nucleotides in length. The selection of thebases for nucleotides in the overhang may be made in an arbitrary manneri.e., the overhang nucleotides may or may not base pair with a targetmRNA. For convenience and simplicity, a two nucleotide overhang isusually a UU overhang (although AA, GG, CC, AC, CA, AG, GA, GC, and CGdi-nucleotide overhangs, and others, are also contemplated, seeVermeulen et al. (2005) RNA 11 (5): 674-682). Preferably, the linkagebetween the nucleotides of the overhang as well as the linkage betweenthe terminal nucleotide of the duplex and the first nucleotide of theoverhang are phosphorothioate linkages. In one particularly preferredembodiment, the antisense strand comprises a UU overhang located at the3′ end of the antisense strand with a phosphorothioate linkage linkingthe 3′ terminal U to the second U nucleotide, and with aphosphorothioate linkage linking the second U nucleotide to the nextnucleotide (in the 5′ direction) in the antisense strand.

In some embodiments, the 5′ end of the sense strand and/or the 3′ end ofthe sense strand and/or the 5′ end of the antisense strand and/or the 3′end of the antisense strand comprises a terminal phosphate. Preferably,a terminal phosphate is located at the 5′ end of the antisense strand.

In some embodiments there are no modified nucleotides (i.e., the 2′position of each of the ribose sugars has an OH moiety). In otherembodiments there are one or more than one chemical modifications. Forexample, there may be one or more or all of:

-   -   (1) 2′-O-alkyl modifications of positions 1 and 2 and all C        nucleotides, and all U nucleotides of the sense strand (e.g.,        O-methyl, O-ethyl, O-n-propyl, O-isopropyl, etc.);    -   (2) a conjugate moiety wherein the conjugate moiety is comprised        of, consists essentially of or consists of a linker and a        conjugate moiety such as a cholesterol moiety and the linker is        attached to the 3′ position of the last nucleotide of the sense        strand;    -   (3) 2′ Fluoro modifications of all C and U nucleotides of the        antisense strand or at least one 2′-O-alkyl modification on the        antisense strand;    -   (4) phosphorylation at the 5′ position of the first nucleotide        of the antisense strand and all other nucleotides may in some        embodiments be unmodified;    -   (5) one or more overhangs; and    -   (6) one or more phosphorothioate modifications associated with        the nucleotides of any overhang on either strand.

In some embodiments, where overhangs are present, 2′-O modifications mayappear in the Cs and Us of the overhangs on the sense strand and2′-fluoro modifications may appear in the Cs and Us of on the antisensestrand. In other embodiments the 2′-O modifications and 2′-fluoro onlyappear within nucleotides in the duplex region. Additionally, in someembodiments it may be desirable to have all of the aforementioned 2′ Csand Us modified in each strand (either including or excluding in anyoverhang regions if present). However, in other embodiments it may bedesirable to have fewer than all of the C and Us on each or eitherstrand contain the aforementioned modifications. When fewer than all Csand Us are modified, the total number of C and U modifications may bechosen by for example, an absolute number, for example 1-8 or 2-7 or 3-6are modified or it may for example be defined in terms of the numberthat are not modified, e.g., all but 1, all but 2, all but 3, all but 4,all but 5, all but 6, all but 7, all but 8 of the Cs or Us areunmodified. In other embodiments, it may be preferable to omit a 2′modification at one or more specific positions, e.g., at one or more ofpositions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,and if present, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. As aperson of ordinary skill in the art will recognize preferably eachstrand contains at least one C or U nucleotide. Additionally, in someembodiments on the antisense strand it may be preferable to have 2′fluoro groups on 0-30 or 0-25 or 0-23 or 0-19 or 1-30 or 1-25 or 1-23 or1-19 or 3-30 or 3-25 or 3-23 or 3-19 or 5-30 or 5-25 or 5-23 or 5-19 or7-30 or 7-25 or 7-23 or 7-19 or 10-30 or 10-25 or 10-23 or 10-19 or 8-15or 10-12 nucleotides. The modified nucleotides may all be pyrimidines,all be purines or be a combination of purines and pyrimidines. In someembodiments all nucleotides on the antisense strand have 2′fluoro groupsand this strand may have at least one pyrimidine, at least one purine,all pyrimidines, all purines or a combination of purines andpyrimidines. Further in some embodiments the 2′fluoro groups are on anyoverhang nucleotides if present while in other embodiments, the overhangnucleotides do not include these modifications. Similarly, in someembodiments on the sense strand it may be preferable to have 2′-O-alkyl(e.g., 2′-O-methyl) groups on 0-30 or 0-25 or 0-23 or 0-19 or 1-30 or1-25 or 1-23 or 1-19 or 3-30 or 3-25 or 3-23 or 3-19 or 5-30 or 5-25 or5-23 or 5-19 or 7-30 or 7-25 or 7-23 or 7-19 or 10-30 or 10-25 or 10-23or 10-19 or 8-15 or 10-12 nucleotides. The modified nucleotides may allbe pyrimidines, all be purines or be a combination of purines andpyrimidines. In some embodiments all nucleotides on the sense strandhave 2′-O-alkyl groups and this strand may have at least one pyrimidine,at least one purine, all pyrimidines, all purines or a combination ofpurines and pyrimidines. Further in some embodiments the 2′-O-alkylgroups are on any overhang nucleotides if present while in otherembodiments, the overhang nucleotides do not include thesemodifications.

In embodiments that have at least one 2′-O-alkyl modification on theantisense strand, there may for example be, from one to ten, one toeight, one to six, one to five, one to four, one to three, or one to twomodifications. In other embodiments, there may be exactly one, two,three, four, five, six, seven, eight, nine or ten such modifications.These at least one modifications may for example be located in a 3′antisense overhang region and/or at one or more of positions one toeight, one to seven, one to six, one to five, one to four, one to three,or one to two of the antisense strand as measured from the 5′ end ofthat strand and within the duplex region.

By way of non-limiting examples, there may be a single 2′-O-alkylmodification (e.g., methyl) at any of positions 1, 2, 3, 4, 5, 6, 7 or 8of the antisense strand. Alternatively, there may be a single 2′-O-alkylmodification in one of the two nucleotides in a UU overhang or in bothof those nucleotides. Other combinations of 2′-O-alkyl modificationsinclude but are not limited to at positions 1 and 2, 1 and 3, 1 and 4, 1and 5, 1 and 6, 1 and 7, 1 and 8, 2 and 3, 2 and 4, 2 and 5, 2 and 6, 2and 7, 2 and 8, 3 and 4, 3 and 5, 3 and 6, 3 and 7, 3 and 8, 4 and 5, 4and 6, 4 and 7, 4 and 8, 5 and 6, 5 and 7, 5 and 8, 6 and 7, 6 and 8, 7and 8, 1 and one of the two nucleotides in a UU overhang or in both ofthose nucleotides, 2 and one of the two nucleotides in a UU overhang orin both of those nucleotides, 3 and one of the two nucleotides in a UUoverhang or in both of those nucleotides, 4 and one of the twonucleotides in a UU overhang or in both of those nucleotides, 5 and oneof the two nucleotides in a UU overhang or in both of those nucleotides,6 and one of the two nucleotides in a UU overhang or in both of thosenucleotides, 7 and one of the two nucleotides in a UU overhang or inboth of those nucleotides, or 8 and one of the two nucleotides in a UUoverhang or in both of those nucleotides.

Furthermore, in some embodiments in which there is at least one2′-O-alkyl modification present on the antisense strand the position isselected such that only A or G bases contain the modification, therebyallowing for all C and U bases to be modified with fluoro groups. Inother embodiments, one or more C or U bases contain the 2′-O-alkylmodification. In those cases, the siRNA may be designed such that any Cand U base that does not have a 2′-O-alkyl modification has a 2′fluoromodification.

In some embodiments, the siRNA contains a duplex region that is 17-30base pairs long or 18-30 base pairs long or 19-30 base pairs long or19-23 base pairs long or 19-21 base pairs long or 18-23 base pairs long.When a duplex region is 17 base pairs long and a 19-mer antisensesequence is provided, it may be that two bases at the 3′ end of theantisense 19-mer form an overhang.

Within the duplex region there may be 100% complementarity or less than100% complementarity, e.g., at least 80% complementarity, at least 85%complementarity, at least 90% complementarity, or at least 95%complementarity. In one embodiment, there is 100% complementarity exceptat sense strand position 6 or at position 13, or at position 19, or atpositions 6 and 13 or at positions 13 and 19 or positions 6 and 19, orat positions 6, 13 and 19. In this example, at the designatedposition(s) there is a mismatch. Mismatches are introduced into theduplex by altering the identity of a nucleotide in the sense strand. Inthis way, the antisense strand retains 100% complementarity with theregion of the target mRNA. Furthermore, as used herein, a positionnumber within a strand refers to the location of that nucleotiderelative to the first, i.e., 5′ most, nucleotide of the duplex region.Thus, position 1 of the sense strand is the 5′ most position of thesense strand, while position 1 of the antisense strand is the 5′ mostposition of the antisense strand. Position 2 is the position immediatelydownstream (or 3′) of position 1 of the respective strand.

As stated above, in some embodiments, a mismatch is introduced into thesense strand. In some cases, the nucleotides introduced at the positionsof mismatch have the same identity or chemical nature as the nucleotidein the antisense strand that normally binds to that particular sensestrand nucleotide. Thus, for example, if one has a double strandedmolecule containing 19 nucleotides in the sense strand and 19nucleotides in the antisense strand with no overhangs on either strand,if a mismatch is introduced at position 6 of the sense strand (countingfrom the 5′ end of the strand), the nucleotide at that position of thesense strand does not pair in a Watson-Crick fashion with the nucleotideat position 14 of the antisense strand. Furthermore, if the nucleotideat position 14 of the antisense strand is e.g., a “C”, then the mismatchwould be achieved by introducing a “C” at position 6 of the sensestrand. As a result of these changes, the nucleotide at position 6 ofthe sense strand no longer has identity with the correspondingnucleotide in the target region of e.g., the mRNA. However, theantisense nucleotide at e.g., position 14 would retain complementarityto the nucleotide on the target region.

The position of the conjugate-linker on the duplex oligonucleotidecomplex can vary with respect to the strand or strands that areconjugated (e.g., the sense strand, the antisense strand, or both thesense and antisense strands), the position or positions within thestrand that are modified (i.e., the nucleotide positions within thestrand or strands), and the position on the nucleotide(s) that aremodified (e.g., the sugar, the base). Conjugate-linkers can be placed onthe 5′ and/or 3′ terminus of one or more of the strands. For example, aconjugate-linker can be placed on the 5′ end of the sense strand and/orthe 3′ end of the sense strand and/or the 3′ end of the antisensestrand. A conjugate-linker can be attached at the 5′ and/or 3′ end of astrand via a phosphodiester bond. In preferred embodiments, aconjugate-linker is attached to the one or both ends of the sense strandvia a phosphodiester bond, more preferably to the 3′ end of the sensestrand.

A conjugate-linker can also be attached to internal positions of thesense strand and/or antisense strand. In addition, multiple positions onthe nucleotides including the 5-position of uridine, 5-position ofcytidine, 4-position of cytidine, 7-position of guanosine, 7-position ofadenosine, 8-position of guanosine, 8-position of adenosine, 6-positionof adenosine, 2′-position of ribose, 5′-position of ribose, and3′-position of ribose, can be employed for attachment of the conjugateto the nucleic acid.

In another embodiment, the present invention provides a method of genesilencing, comprising introducing into a cell in vitro at least onesiRNA that comprises a sequence that is selected from SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, and 88. The siRNA can be introduced by allowingpassive uptake of siRNA, or through the use of a vector.

Any of the methods and kits disclosed herein can employ eitherunimolecular siRNAs, siRNAs comprised of two separate polynucleotidestrands, or combinations thereof. Furthermore, any of the methodsdisclosed herein can be used in gene silencing using a variety ofdifferent protocols. In one non-limiting example, two or more siRNAstargeting the same gene can be administered simultaneously. As is thecase with individual siRNAs, the two or more siRNA can be administeredin a single dose or single transfection, in multiple doses, or as thecase may be.

In one embodiment the invention provides the use of a compound thatinhibits the expression and/or activity of a VEGFA gene for themanufacture of a medicament for treatment of a disorder associated withover-expression of VEGFA. The medicaments may, for example, beadministered orally, parenterally (including subcutaneously,intramuscularly, or intravenously), rectally, transdermally, buccally,or nasally. The medicaments may comprise any one or more of thecompounds described herein.

Interfering RNA may be delivered directly to the eye by ocular tissueinjection such as periocular, conjunctival, subtenon, intracameral,intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, orintracanalicular injections; by direct application to the eye using acatheter or other placement device such as a retinal pellet, intraocularinsert, suppository or an implant comprising a porous, non-porous, orgelatinous material; by topical ocular drops or ointments; or by a slowrelease device in the cul-de-sac or implanted adjacent to the sclera(transscleral) or in the sclera (intrascleral) or within the eye.Intracameral injection may be through the cornea into the anteriorchamber to allow the agent to reach the trabecular meshwork.Intracanalicular injection may be into the venous collector channelsdraining Schlemm's canal or into Schlemm's canal.

For ophthalmic delivery, an interfering RNA may be combined withophthalmologically acceptable preservatives, co-solvents, surfactants,viscosity enhancers, penetration enhancers, buffers, sodium chloride, orwater to form an aqueous, sterile ophthalmic suspension or solution.Solution formulations may be prepared by dissolving the interfering RNAin a physiologically acceptable isotonic aqueous buffer. Further, thesolution may include an acceptable surfactant to assist in dissolvingthe interfering RNA. Viscosity building agents, such as hydroxymethylcellulose, hydroxyethyl cellulose, methylcellulose,polyvinylpyrrolidone, or the like may be added to the compositions ofthe present invention to improve the retention of the compound.

In order to prepare a sterile ophthalmic ointment formulation, theinterfering RNA is combined with a preservative in an appropriatevehicle, such as mineral oil, liquid lanolin, or white petrolatum.Sterile ophthalmic gel formulations may be prepared by suspending theinterfering RNA in a hydrophilic base prepared from the combination of,for example, CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like,according to methods known in the art. VISCOAT® (Alcon Laboratories,Inc., Fort Worth, Tex.) may be used for intraocular injection, forexample. Other compositions of the present invention may containpenetration enhancing agents such as cremephor and TWEEN® 80(polyoxyethylene sorbitan monolaureate, Sigma Aldrich, St. Louis, Mo.),in the event the interfering RNA is less penetrating in the eye.

The present invention also provides pharmaceutical compositions thatcomprise an siRNA of the present invention in a pharmaceuticallyacceptable carrier. Thus, in another embodiment, the present inventionis directed to a pharmaceutical composition comprising a therapeuticallyeffective amount of an siRNA, wherein the siRNA consists of: (a) a sensestrand and an antisense strand that form a duplex region, wherein theduplex region is 17-30 base pairs in length and comprises an antisenseregion that has a sequence selected from the group consisting of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86 and 88; and (b) a sense region that is 100%complementary to the antisense region or has mismatches at one or morepositions. In one example, the molecule consists of a sense strand andan antisense strand that form a 19 base double stranded complex andmismatches that are located at positions 6, 13 or 19 of the senseregion, wherein said positions are defined relative to the 5′ mostnucleotide of the sense strand that is part of the duplex region. Forall of the descriptions relayed above, the following modifications canbe adopted: sense region positions 1 and 2 and all Cs and Us have2′-O-Me modifications, and all other 2′ positions of the sense regionhave 2′-OH groups, and wherein all Cs and Us of the antisense region are2′-F modified, all other nucleotides of the antisense region have 2′-OHgroups, and the nucleotide at position 1 of the antisense region isphosphorylated and there is a UU overhang attached to the 3′ end of theantisense region, wherein the internucleotide bond between the twonucleotides of the overhang as well as the first nucleotide of theoverhang and the 3′ most antisense nucleotide of the duplexed region ofthe antisense strand is a phosphorothioate linkage; and a cholesterolmoiety is attached to the 3′ end of the sense region by a C5 linker. Inyet another embodiment, the siRNA has the same features as theaforementioned but the antisense strand has at least one 2′-O-Memodification instead of a 2′-F modification.

The pharmaceutically acceptable carrier may comprise one or more ofexcipients, such as vehicles adjuvants, pH adjusting and bufferingagents, tonicity adjusting agents, stabilizers and wetting agents.Furthermore, in some embodiments, the siRNA is delivered inmicrocapsules, for example by coacervation techniques or by interfacialpolymerization (e.g., hydroxymethylcellulose or gelatin-microcapsulesand poly-(methylmethasylate) microcapsules, respectively) in colloidaldrug delivery systems (for example, liposomes, microspheres,microemulsions, nano-particles, and nanocapsules or microemulsions).

The siRNA may be introduced into a cell or organism by any method thatis now known or that comes to be known and that from reading thisdisclosure, persons skilled in the art would determine would be usefulin connection with the present invention in enabling siRNA to cross thecellular membrane. These methods include, but are not limited to, anymanner of transfection, such as, for example, transfection employingDEAE-Dextran, calcium phosphate, cationic lipids/liposomes, micelles,manipulation of pressure, microinjection, electroporation,immunoporation, use of vectors such as viruses, plasmids, cosmids,bacteriophages, cell fusions, and coupling of the polynucleotides tospecific conjugates or ligands such as antibodies, antigens, orreceptors, passive introduction, adding moieties to the siRNA thatfacilitate its uptake, and the like.

In another embodiment, the present invention features use of an siRNAthat targets VEGFA in the manufacture of a medicament for treating,inhibiting or ameliorating one or more of the following conditions:psoriasis, cancer, rheumatoid arthritis, ocular neovascularization,abnormal angiogenesis, retinal vascular permeability, retinal edema,diabetic retinopathy (particularly proliferative diabetic retinopathy),diabetic macular edema, exudative age-related macular degeneration,sequela associated with retinal ischemia, and posterior segmentneovascularization. Recipients of the siRNAs of the present inventionmay for example, be persons who are afflicted with one or more of theaforementioned disorders.

The dosage of the siRNA is preferably a therapeutically effectiveamount. A therapeutically effective amount will be determined at leastin part by the age, weight and condition or severity of the affliction othe organism to be treated.

Examples of the siRNAs of the present invention may comprise anantisense sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and88, and the corresponding sense strand in Table I.

Table 1 shows the silencing activities of 42 unmodified siRNAs tested invitro as described in the Examples Section.

% VEGFA % VEGFA SEQ ID Top: Sense Strand, 5′→3′ RNA Protein Seq. Ref.NO: Bottom: Antisense strand, 5′→3′ remaining remaining vegfa 2.1 1UACUAAAUCUCUCUCCUUU 41.6 50.0 2 AAAGGAGAGAGAUUUAGUA vegfa 2.2 3ACAGAACGAUCGAUACAGA 22.5 20.6 4 UCUGUAUCGAUCGUUCUGU vegfa 2.3 5CGACAGAACAGUCCUUAAU 19.9 21.2 6 AUUAAGGACUGUUCUGUCG vegfa 2.4 7GAAGAGACACAUUGUUGGA 22.3 21.4 8 UCCAACAAUGUGUCUCUUC vegfa 2.5 9GUCACUAGCUUAUCUUGAA 13.6 31.6 10 UUCAAGAUAAGCUAGUGAC vegfa 2.6 11CAGCACACAUUCCUUUGAA 57.3 44.4 12 UUCAAAGGAAUGUGUGCUG vegfa 2.9 13GGAGACCACUGGCAGAUGU 24.1 37.5 14 ACAUCUGCCAGUGGUCUCC VEGFA 2.11 15GCUCGGUGCUGGAAUUUGA 51.0 35.1 16 UCAAAUUCCAGCACCGAGC vegfa 2.12 17GAAAGACAGAUCACAGGUA 20.8 27.8 18 UACCUGUGAUCUGUCUUUC vegfa 2.14 19CCAGAAACCUGAAAUGAAG 31 22.5 20 CUUCAUUUCAGGUUUCUGG vegfa 2.15 21GAGAAGAGACACAUUGUUG 36.6 20.5 22 CAACAAUGUGUCUCUUCUC vegfa 2.17 23CGACAAAGAAAUACAGAUA 40.8 40.0 24 UAUCUGUAUUUCUUUGUCG vegfa 2.18 25GGGCAAAUAUGACCCAGUU 12.2 39.8 26 AACUGGGUCAUAUUUGCCC vegfa 2.19 27GAAGAGAAGAGACACAUUG 43.3 21 28 CAAUGUGUCUCUUCUCUUC vegfa 2.20 29GAAACCAGCAGAAAGAGGA 47.6 44 30 UCCUCUUUCUGCUGGUUUC vegfa 2.21 31GAUCACAGGUACAGGGAUG 44.8 33.1 32 CAUCCCUGUACCUGUGAUC vegfa 2.22 33GGAAAGAGGUAGCAAGAGC 52 53.3 34 GCUCUUGCUACCUCUUUCC vegfa 2.23 35GAGAUGAGCUUCCUACAGC 88.5 14.7 36 GCUGUAGGAAGCUCAUCUC vegfa 2.24 37GAUCAAACCUCACCAAGGC 30.8 11.3 38 GCCUUGGUGAGGUUUGAUC Vegfa 2.25 39CAACAAAUGUGAAUGCAGA 22.0 5.52 40 UCUGCAUUCACAUUUGUUG vegfa 3.1 41AAAUGAAGGAAGAGGAGAC 16.2 9 42 GUCUCCUCUUCCUUCAUUU vegfa 3.2 43AAUGCAGACCAAAGAAAGA 25.3 11.5 44 UCUUUCUUUGGUCUGCAUU vegfa 3.3 45ACAUAGGAGAGAUGAGCUU 16.3 14.3 46 AAGCUCAUCUCUCCUAUGU vegfa 3.4 47ACGACAAAGAAAUACAGAU 32.6 52.4 48 AUCUGUAUUUCUUUGUCGU vegfa 3.5 49AGACACACCCACCCACAUA 17.6 26.1 50 UAUGUGGGUGGGUGUGUCU vegfa 3.6 51AGACAUUGCUAUUCUGUUU 31.4 25.7 52 AAACAGAAUAGCAAUGUCU vegfa 3.7 53AGAGAAAAGAGAAAGUGUU 23.4 9.8 54 AACACUUUCUCUUUUCUCU vegfa 3.8 55AGCACACAUUCCUUUGAAA 26.6 42 56 UUUCAAAGGAAUGUGUGCU vegfa 3.9 57CAAAUGUGAAUGCAGACCA 45.8 35.4 58 UGGUCUGCAUUCACAUUUG vegfa 3.10 59CACACAUUCCUUUGAAAUA 39.3 28.3 60 UAUUUCAAAGGAAUGUGUG vegfa 3.11 61CAGAACAGUCCUUAAUCCA 22.9 34.4 62 UGGAUUAAGGACUGUUCUG vegfa 3.12 63CAGAGAAAAGAGAAAGUGU 30.1 30.7 64 ACACUUUCUCUUUUCUCUG vegfa 3.13 65CCAGCACAUAGGAGAGAUG 34.7 22.3 66 CAUCUCUCCUAUGUGCUGG vegfa 3.16 67CGAGAUAUUCCGUAGUACA 32.8 61 68 UGUACUACGGAAUAUCUCG vegfa 3.17 69CUACUGUUUAUCCGUAAUA 40.9 55.2 70 UAUUACGGAUAAACAGUAG vegfa 3.18 71CUGAAAUGAAGGAAGAGGA 43.5 43 72 UCCUCUUCCUUCAUUUCAG vegfa 3.19 73GAAAUGAAGGAAGAGGAGA 42.7 48.3 74 UCUCCUCUUCCUUCAUUUC vegfa 3.20 75GAACAGUCCUUAAUCCAGA 25.9 30.9 76 UCUGGAUUAAGGACUGUUC vegfa 3.21 77GAGAGAUGAGCUUCCUACA 64.6 33 78 UGUAGGAAGCUCAUCUCUC vegfa 3.22 79GAGAUAUUCCGUAGUACAU 60.5 61.3 80 AUGUACUACGGAAUAUCUC vegfa 3.23 81GAGGCAGAGAAAAGAGAAA 26.9 32.4 82 UUUCUCUUUUCUCUGCCUC vegfa 3.24 83GAUAUUAACAUCACGUCUU 37.8 73.5 84 AAGACGUGAUGUUAAUAUC vegfa 3.25 85GCACACAUUCCUUUGAAAU 18.8 38.1 86 AUUUCAAAGGAAUGUGUGC vegfa 3.26 87GCGGAUCAAACCUCACCAA 30 33.5 88 UUGGUGAGGUUUGAUCCGC

Table 2 shows the silencing activity of a selection of siRNAs inunmodified and siStable modified formats. Specifically, data in columnsD and E are derived from unmodified molecules at 24 hours. Data incolumns F, G, H, and I are derived from siStable modified molecules at72 hours. Data in columns F and G were derived when siRNA weretransfected into cells at 100 nM concentrations.

B D E F G H I A SEQ C RNA protein siStable siStable RNA protein Seq. IDTop: Sense Strand, 5′→3′ IC₅₀ IC₅₀ % RNA % protein IC₅₀ IC₅₀ Ref. NO:Bottom: Antisense strand, 5′→3′ (nM) (nM) remaining remaining (nM) (nM)vegfa 1 UACUAAAUCUCUCUCCUUU 0.31 0.51 17 41 4.78 2.35 2.1 2AAAGGAGAGAGAUUUAGUA vegfa 41 AAAUGAAGGAAGAGGAGAC 5.2 0.82 18 12 0.360.15 3.1 42 GUCUCCUCUUCCUUCAUUU vegfa 43 AAUGCAGACCAAAGAAAGA 0.93 0.1710 9 0.4 0.37 3.2 44 UCUUUCUUUGGUCUGCAUU vegfa 45 ACAUAGGAGAGAUGAGCUU0.43 0.59 11 10 0.82 0.77 3.3 46 AAGCUCAUCUCUCCUAUGU vegfa 51AGACAUUGCUAUUCUGUUU 0.81 0.94 28 31 23 3.17 3.6 52 AAACAGAAUAGCAAUGUCUvegfa 53 AGAGAAAAGAGAAAGUGUU 1.3 0.46 10 10 0.26 0.46 3.7 54AACACUUUCUCUUUUCUCU vegfa 59 CACACAUUCCUUUGAAAUA 1.05 0.97 13 20 0.260.17 3.10 60 UAUUUCAAAGGAAUGUGUG vegfa 63 CAGAGAAAAGAGAAAGUGU 4.6 1 1015 0.11 0.21 3.12 64 ACACUUUCUCUUUUCUCUG vegfa 75 GAACAGUCCUUAAUCCAGA 20.62 9 28 0.31 0.53 3.20 76 UCUGGAUUAAGGACUGUUC vegfa 81GAGGCAGAGAAAAGAGAAA 1.4 1.2 14 45 2.2 2.7 3.23 82 UUUCUCUUUUCUCUGCCUC

It is noted that the above recited duplexes do not contain mismatches.The present invention includes the specifically recited siRNAs as wellas pharmaceutical compositions that contain them and methods for usingthem. The present invention also includes siRNAs that are similar tothem but have a different base at position 6 or position 13 or position19 of the sense strand or at both positions 6 and 13 or both ofpositions 13 and 19 or at both of positions 6 and 19 of the sense strandor at all three of positions, 6, 13 and 19 of the sense strand. Thus atany of those three positions, wherein in tables 1 or 2 there is an Acomplementary to a U, a U, C or G may be inserted, wherein in tables 1or 2 there is an U complementary to an A, an A, C or G may be inserted,wherein in tables 1 or 2 there is a C complementary to a G, a U, A or Gmay be inserted, wherein in tables 1 or 2 there is a G complementary toa C, a U, C or A may be inserted. Still further, any of these siRNAs maycontain overhang regions, e.g., a UU 3′ antisense overhang and/or a UU3′ sense overhang.

By way of further example, in one embodiment, the present invention isdirected to an siRNA from Table 2, or to an siRNA that differs from thatof table 2 in that the sense strand has three mismatched nucleotidesthat are located at positions, 6, 13, and 19 with the oppositenucleotides on the antisense e.g., an siRNA that contains the sense andantisense sequences of vegfa 3.7, except that the sense strand has threemismatched nucleotides that are located at positions, 6, 13, and 19. Insome embodiments the mismatches are selected such that one or more, forexample, two or three of the mismatched bases are the same as the baseson the opposite strand and no other mismatched bases are present in theduplex. By way of a non-limiting example, for vegfa 3.7 a duplex may be

SS- 5′ AGAGAUAAGAGAUAGUGUA 3′ (SEQ ID No: 91) AS- 3′UCUCUUUUCUCUUUCACAA 5′ (SEQ ID No: 54)

This duplex, as well as any other duplex disclosed herein, may contain3′ overhangs on either the sense strand or the antisense strand. By wayof a non-limiting example, there may be a dinucleotide overhangs, e.g.,UU. This overhang may exist on the sense strand, but not the antisensestrand; on the antisense strand but not the sense strand; on bothstrands or on neither strand. Each overhang may be constructed to have astandard internucleotide linkage between nucleotides of the overhang anda standard linkage to the 3′ end of the appropriate strand of theduplex, or in the overhang, the bond between the two nucleotides of theoverhang as well as the first nucleotide of the overhang and the 3′ mostantisense nucleotide of the duplexed region of the strand is aphosphorothioate linkage. Thus, e.g., in the vegfa 3.7 duplex of thepreceding paragraph, SEQ ID No: 54 may contain a UU 3′ antisenseoverhang that does not contain a phosphorothioate linkage between thenucleotides of the overhang or between the overhang and the 3′ of SEQ IDNo: 54, or there may be phosphorothioate linkages at one or both ofthose positions.

Unless otherwise specified, each of the features of each of theaforementioned embodiments may be used in connection with any of theother embodiments, unless such use is incompatible or inconsistent withthat embodiment.

Having described the invention with a degree of particularity, exampleswill now be provided. These examples are not intended to and should notbe construed to limit the scope of the claims in any way.

EXAMPLES Example 1 General Techniques for in Vitro Studies

siRNA Selection for Study

A collection of siRNAs capable of targeting all the variants of VEGFAwere identified (NM_(—)001025366, NM_(—)003376, NM_(—)001025367,NM_(—)001025368, NM_(—)001033756, NM_(—)001025369, NM_(—)001025370).Table 1 provides a list of the siRNAs along with the sense and antisensestrand sequences (5′→3′).

To assess the relative functionality of each siRNA, sequences weresynthesized using 2′ ACE chemistry (U.S. Pat. No. 6,008,400; U.S. Pat.No. 6,111,086; U.S. Pat. No. 6,590,093; Scaringe (2000) Methods inEnzymology 317:3-18; Scaringe (2001) Methods 23(3):206-217) and thentransfected into HeLa cells (ATCC, #CCL-2) by lipid mediatedtransfection using the manufacturer's protocols (10,000 cells per wellin a 96 well format, 100 nM siRNA, 0.2 μl DharmaFECT 1/well).Seventy-two hours post-transfection, overall cell viability and targetknockdown at the mRNA and protein level was determined. All assays wereperformed in triplicate and for a select group of siRNAs, a dose curve(0.001, 0.01, 0.1, 1.0, 10.0, and 100 nM) was performed to ascertain theIC₅₀ for the siRNA/target mRNA pair. Positive and negative controls wereincluded in all experiments and consisted of a non-targeting control(NTC #5 sense strand sequence: 5′-UGGUUUACAUGUCGACUAAUU-3′ (SEQ ID NO:89)) and a positive control targeting PPIB (sense strand sequence:5′-ACAGCAAAUUCCAUCGUGU-3′ (SEQ ID NO: 90)). Note: the positive controlmolecule used in these studies contains the following modifications:sense strand contains a 2′-O-methyl modification on the first twonucleotides counting from the 5′ end of the strand; antisense strandcontains a 5′ phosphate group; both sense and antisense strands containa 2 nucleotide UU overhang on the 3′ end.

Target mRNA and Protein Knockdown Analysis

Target mRNA knockdown was determined at 72 hour post-transfection usingthe branched DNA assay (QuantiGene Screen Kit, Panomics). The expressionof PPIB was used as a reference mRNA and the targeted mRNA knockdown wasfurther normalized to the corresponding non-targeting control (NTC).Protein expression was assessed by performing a VEGFA ELISA assay onsupernatants from transfected cells at 72 hours post-transfection. TheELISA was performed according to the manufacturer's instructions using50 μL of supernatant (Human VEGFA ELISA kit, Thermo Scientific).Absorbance was read on a spectrophotometer at 450 nM. Data wasnormalized to the corresponding NTC control.

Cell Viability Assay

Cell viability was assessed by a resazurin assay at 72 hourspost-transfection. Resazurin was added directly into the culture mediaand the plates were incubated for 1-1.5 hours prior to measuring thefluorescence on a Wallac VICTOR 2 (Perkin Elmer Life Sciences) platereader (Excitation 530 nm, Emission_(—)590 nm and 1 second exposure).Data was normalized to the corresponding NTC control.

siRNA Designs for Study

siRNA configurations tested in the in vitro studies include (1) thestandard unmodified design (19 base pairs duplex, UU overhangs on the 3′end of both sense and antisense strands), and (2) the stabilized design(a 19 base pair duplex; sense strand modifications: 2′-O-methylmodifications on nucleotides 1 and 2 (counting from the 5′ end of thestrand) plus 2′-O-methyl modifications on all Cs and Us; antisensestrand modifications: a phosphate on the 5′ terminal nucleotide, 2′ Fmodifications on all Cs and Us, a 2 nucleotide (UU) overhang on the 3′terminus, and a phosphorothioate internucleotide modification betweenthe two nucleotides of the overhang and between the first (3′ most)nucleotide of the duplex and the first nucleotide of the overhang).

For in vivo studies, siRNAs included the following design:

-   -   a 19 bp duplex    -   sense strand modifications        -   2′-O-methyl modifications on nucleotides 1 and 2 (counting            from the 5′ end of the strand)        -   2′-O-methyl modifications on all Cs and Us        -   cholesterol conjugated to the 3′ terminus using a C5 linker            (see U.S. Pat. Pub. 2009/0209626, published Aug. 20, 2009            the disclosure of which is incorporated by reference as if            set forth fully herein)    -   antisense strand modifications        -   5′ phosphate        -   2′ F on all Cs and Us        -   a two nucleotide (UU) overhang on the 3′ terminus        -   phosphorothioate internucleotide modifications between the            two nucleotides of the overhang and between the first (3′            most) nucleotide of the duplex and the first nucleotide of            the overhang.

In addition, mismatches at positions 6, 13, and 19 have beenincorporated into molecules used in in vivo studies. In all cases,mismatches between the two strands of the siRNA are achieved by changingthe nucleotide of the sense strand to have identity with the base (onthe antisense strand) that typically pairs with that position. Thus, forinstance, if the sense-antisense pair at sense strand position 6 isnormally U-A, then the mismatch will be introduced by converting thepair to A-A. Similarly, if the sense-antisense pair at sense strandposition 6 is G-C, then the mismatch will be C-C. In this way, amismatch is incorporated into the duplex, but the antisense strandremains the reverse complement of the intended target.

Example 2 Results of In Vitro and In Vivo Studies

The performance of all the sequences tested in vitro is shown inTable 1. Multiple sequences were observed to provide greater than 70%gene knockdown at both the RNA and protein level including, forinstance, Vegfa 2.2, 2.3, 2.4, 2.12, 3.1, 3.2, 3.3, 3.5, and 3.7. Inaddition, when a subset of the collection was tested with the stabilizeddesign, overall performance was found to be equivalent or better thanthat observed in the unmodified state (see, for instance, vegfa 2.1,3.2, 3.3). As Table 2 shows, in both the unmodified and modified states,IC₅₀ for RNA knockdown ranged from approximately 0.11→23 nM while IC₅₀for protein knockdown ranged from ˜0.17→3.17 nM. Based on these results,two sequences, Vegfa 3.2 and 3.7, were re-synthesized using the in vivodesign (referred to as “Accell”) described previously. The results ofthese experiments may be further demonstrated by reference to theaccompanying figures.

FIGS. 1A and 1B illustrate the effect of intravitreal (IVT) injection ofAccell VEGFA siRNAs on expression of VEGFA mRNA and protein,respectively, in the rat retina at 72 h post-injection. Lewis ratsreceived 10 μg IVT injections (OD) of Accell VEGFA 3.2, Accell VEGFA 3.7or Accell non-targeting control #1 (NTC1) siRNAs resuspended in 1× siRNAbuffer (Dharmacon). The Accell NTC1 siRNA sense strand sequence is5′-UGGUUAACAUGUCGACUAA-3′ (SEQ ID NO: 92); the Accell NTC1 siRNAantisense strand sequence is 5′-UUAGUCGACAUGUAAACCAUU-3′ (SEQ ID NO:93). Contralateral eyes (OS) were not treated. Eyes were harvested at 72h post-injection, and retinas were isolated by dissection. (1A) TotalRNA was extracted using Trizol (Invitrogen), and VEGFA and β-actin(ACTB) mRNA levels were determined by Taqman qRT-PCR assay (AppliedBiosystems). VEGFA mRNA expression was normalized to β-actin expression.(1B) Protein was extracted using RIPA buffer (Pierce), and rat VEGFAprotein level was determined by ELISA (R&D Systems). VEGFA proteinexpression was normalized to total protein determined by BCA assay(Pierce). Data are presented as the mean (n=6)±standard deviation (errorbars). *, P<0.001 versus NTC1. Both of the VEGFA siRNAs significantlyreduced the expression of VEGFA mRNA; VEGFA 3.7 also significantlyreduced VEGFA and protein. The NTC1 control siRNA had little, if any,effect on VEGFA expression.

FIG. 2 shows a comparison between the dose response curves for AccellVEGFA 3.2 siRNA and a control siRNA in the rat retina. Lewis ratsreceived 1-25 μg IVT injections (OD) of Accell VEGFA 3.2 or Accell NTC1control siRNAs resuspended in 1× siRNA buffer (Dharmacon). Contralateraleyes (OS) were not treated. Eyes were harvested at 72 h post-injection,and retinas were isolated by dissection. Total RNA was extracted usingTrizol Plus (Invitrogen), and VEGFA and β-actin mRNA levels weredetermined by Taqman qRT-PCR assay (Applied Biosystems). VEGFA mRNAexpression was normalized to β-actin mRNA. Data are presented as themean OD:OS ratio (ratio of VEGFA level in the treated eye versus thenon-treated eye) for normalized VEGFA mRNA expression (n=6)±standarddeviation (error bars). *, P<0.05; **, P<0.01 versus Accell NTC1.Intravitreal injection of increasing amounts of Accell VEGFA siRNA 3.2resulted in a dose response that reached essentially complete silencingof VEGFA mRNA expression at 25 μg.

FIGS. 3A and 3B shows a comparison between the dose response curves forAccell VEGFA 3.7 siRNA and a control siRNA in the rat retina. Lewis ratsreceived 1-50 μg IVT injections (OD) of Accell VEGFA 3.7 siRNA or AccellVEGFA 3.7 cleavage site mismatch (CS MM) control siRNAs resuspended in1× siRNA buffer (Dharmacon). The Accell VEGFA 3.7 CS MM control siRNAhas the same sequence as Accell VEGFA 3.7 siRNA except for a3-nucleotide mismatch to the VEGFA mRNA target sequence. The AccellVEGFA 3.7 CS MM siRNA sense strand sequence is 5′-AGAGAUAACUCAUAGUGUA-3′(SEQ ID NO: 94); the Accell VEGFA 3.7 CS MM siRNA antisense strandsequence is 5′-AACACUUUGAGUUUUCUCUUU-3′ (SEQ ID NO: 95). Contralateraleyes (OS) were not treated. Eyes were harvested at 72 h post-injection,and retinas were isolated by dissection. (3A) Total RNA was extractedusing Trizol (Invitrogen), and VEGFA and β-actin mRNA levels weredetermined by Taqman qRT-PCR assay (Applied Biosystems). VEGFA mRNAexpression was normalized to β-actin mRNA expression. (3B) Protein wasextracted using RIPA buffer (Pierce), and rat VEGF-A level wasdetermined by ELISA (R&D Systems). VEGFA protein expression wasnormalized to total protein level determined by BCA assay (Pierce). Dataare presented as the mean OD:OS ratio for normalized VEGFA mRNA orprotein expression (n=6)±standard deviation (error bars). *, P<0.03; **,P<0.0002; #, P<0.005. Intravitreal injection Accell VEGFA 3.7 siRNA atdoses as low as 5 μg caused a significant reduction in VEGFA expressionat both the mRNA and protein levels. The Accell VEGFA 3.7 siRNAexhibited a dose response that reached >70% inhibition of VEGFA mRNAexpression and approximately 80% inhibition of VEGFA protein expressionat 25 μg siRNA. Non-RNAi-mediated inhibition of VEGFA expression wasalso observed with the control siRNA. This effect was less pronouncedfor VEGFA protein than for VEGFA mRNA.

FIGS. 4A and 4B show the time duration of action for the Accell VEGFA3.7 siRNA. Lewis rats received 25 μg IVT injections (OD) of Accell VEGFA3.7 siRNA or Accell NTC1 control siRNAs resuspended in 1× siRNA buffer(Dharmacon). Contralateral eyes (OS) were not treated. Eyes wereharvested at 1, 3, 7, 14, 28, 42, and 56 d post-injection, and retinaswere isolated by dissection. Expression of VEGFA mRNA (4A) and VEGFAprotein (4B) was evaluated as described in the previous examples. Dataare presented as the mean OD:OS ratio for normalized VEGFA mRNA orprotein expression (n=6)±standard deviation (error bars). *, P<0.001; #,P<0.002 versus NTC1. Intravitreal injection of Accell VEGFA 3.7 siRNAcaused significant inhibition of VEGFA mRNA and protein expressionwithin 24 h. Inhibition persisted for several weeks.

FIGS. 5A and 5B show inhibition of VEGFA protein expression andpreretinal neovascularization, respectively, in the rat oxygen-inducedretinopathy (OIR) model (modified from Penn et al., Pediatr. Res.36:724-731, 1994). Following 14 d of cycling between 50% and 10% O₂,neonatal Sprague Dawley rats were exposed to room air (21% O₂) for 7 d(postpartum days 15-21, P15-P21). On days P15 and P18, animals received25 μg IVT injections (OS) of Accell VEGFA siRNA 3.7 or Accell VEGFA 3.7CS MM control siRNA resuspended in 1× siRNA buffer (Dharmacon).Contralateral eyes (OD) were treated with vehicle (1× siRNA buffer).Injection volume was 1 μl. Eyes were harvested on day P21, and retinaswere isolated by dissection. (5A) Protein was extracted using RIPAbuffer (Pierce), and VEGFA protein level was determined by ELISA (R&DSystems). VEGFA protein expression was normalized to total protein leveldetermined by BCA assay (Pierce). Data are presented as mean normalizedVEGFA protein expression (n=7)±standard deviation (error bars). *,P<0.03. (5B) Retinas were fixed in 10% neutral buffered formalin for 24h, subjected to ADPase staining, and fixed onto slides as whole mounts.Images were acquired using a Nikon Coolscope®, and each of 12 sectorsper retina was assessed for the presence or absence ofneovascularization to obtain a clockhour score (n=6-8). #, P<0.05. TheAccell VEGFA 3.7 siRNA caused a significant reduction in VEGFA proteinexpression (˜40%), resulting in an approximately 88% inhibition ofpreretinal neovascularization. The Accell VEGFA 3.7 CS MM control siRNAdid not have a significant effect on either VEGFA expression orneovascularization.

As persons of ordinary skill in the art are aware, extrapolating tohumans, observations made in rats is well-known.

1. A method for decreasing expression of VEGFA in vivo, comprisingadministering an siRNA to a subject, wherein the siRNA comprises asequence that is a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, and
 88. 2. The method according to claim 1, wherein the subjectis a mammal.
 3. The method according to claim 2, wherein the subject isa human.
 4. The method according to claim 1, wherein the siRNA consistsof a duplex region that is 17-30 base pairs in length and either nooverhang regions, one overhang region or two overhang regions, whereineach overhang region has 6 or fewer bases.
 5. The method according toclaim 1, wherein the siRNA is a shRNA that has a duplex region that is17-30 base pairs in length.
 6. The method according to claim 1, wherein,the siRNA comprises: (a) a sense strand; and (b) an antisense strand,wherein each of said sense strand and said antisense strand is 19-36nucleotides in length and the antisense strand comprises a sequence thatis selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and
 88. 7. Themethod according to claim 6, wherein: (a) within the sense strand,nucleotides at positions 1 and 2, all C nucleotides and all Unucleotides have 2′-O-methyl modifications, and all other nucleotides ofthe sense strand have 2′OH groups; and (b) within the antisense strand,all C nucleotides and all U nucleotides have 2′ Fluoro modifications andall other nucleotides of the antisense strand have 2′OH groups, and thenucleotide at position 1 of the antisense strand is 5′ phosphorylated.8. The method according to claim 7, wherein the two 3′ most bases of theantisense strand are UU.
 9. The method according to claim 8, wherein theantisense strand is twenty-one bases in length.
 10. The method accordingto claim 9, wherein the sense strand is nineteen bases in length and theantisense strand and the sense strand form a duplex region that isnineteen base pairs in length.
 11. The method according to claim 6,wherein the siRNA further comprises a cholesterol moiety attached to the3′ end of the sense strand and the cholesterol moiety is attached by aC5 linker, and wherein the duplex region contains mismatches atpositions 6, 13 and 19 of the sense strand, wherein said positions aredefined relative to the 5′ end of the sense strand, and the sense stranddoes not contain a 5′ overhang region.
 12. The method according to claim7, wherein the siRNA further comprises a cholesterol moiety attached tothe 3′ end of the sense strand and the cholesterol moiety is attached bya C5 linker, and wherein the duplex region contains mismatches atpositions 6, 13 and 19 of the sense strand, wherein said positions aredefined relative to the 5′ end of the sense strand, and the sense stranddoes not contain a 5′ overhang region.
 13. The method according to claim12, wherein the antisense strand is twenty-one bases in length.
 14. Themethod according to claim 13, wherein the sense strand is nineteen basesin length and the antisense strand and the sense strand form a duplexregion that is nineteen base pairs in length.
 15. The method accordingto claim 14, wherein the antisense strand comprises SEQ ID NO: 54 16.The method of claim 15, wherein within the sense strand said mismatchednucleotides at positions, 6, 13, and 19 are the same base as theopposite nucleotides on the antisense.
 17. A pharmaceutical compositioncomprising a therapeutically effective amount of an siRNA, wherein thesiRNA comprises: (a) an antisense strand that is nineteen to thirty-sixbases in length and that has a sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88 (b) a sensestrand that is nineteen to thirty-six bases in length, wherein theantisense strand and the sense strand form a duplex region of seventeento thirty base pairs and within the duplex region are at least 75%complementary.
 18. The pharmaceutical composition of claim 17, whereinwithin the duplex region, there are mismatches at one or more ofpositions 6, 13 or 19, wherein said positions are defined relative tothe 5′ end of the sense strand.
 19. The pharmaceutical composition ofclaim 18, wherein within the sense strand positions 1 and 2 and all Csand Us have 2′-OMe modifications, and all other positions of the sensestrand have 2′-OH groups, and wherein all Cs and Us of the antisensestrand have 2′-F modifications, and all other nucleotides of theantisense strand have 2′-OH groups, and the nucleotide at position 1 ofthe antisense region is phosphorylated.
 20. The pharmaceuticalcomposition of claim 19, wherein the sense strand is nineteen bases inlength and the antisense strand is twenty-one bases in length and thetwo 3′ most bases of the antisense strand are UU overhang.
 21. Thepharmaceutical composition of claim 20, wherein a cholesterol moiety isattached to the 3′ end of the sense region by a C5 linker.